1. To clarify the mechanism by which cytosolic free Mg2+ concentrations ([Mg2+]i) are regulated in human platelets, we investigated platelet membrane permeability to Mg2+ by altering extracellular Mg2+ concentrations, and tested the existence of a Na(+)-Mg2+ exchanger by manipulating the transmembrane Na+ gradient. 2. Platelet [Mg2+]i was 421 +/- 52 mumol/l in healthy men. [Mg2+]i remained constant during a 120 min exposure to nominally zero (low) or 5 mmol/l (high) external Mg2+ concentrations. 3. Preincubation of platelets with 10(-4) mol/l ouabain effectively decreased the transmembrane Na+ gradient. The ouabain-induced increase in [Mg2+]i was statistically significant after 30 min of exposure (14.6 +/- 2.0%) and reached 24.6 +/- 4.5% after 60 min. Similarly, the replacement of extracellular Na+ with N-methyl-D-glucamine significantly increased [Mg2+]i by 48.5 +/- 3.9% and 78.8 +/- 12.5%, respectively. 4. These results suggest that [Mg2+]i is well controlled in the presence of large transmembrane Mg2+ concentration gradients, and a Na(+)-Mg2+ exchanger may be involved in the regulation of [Mg2+]i in human platelets.